Compositions for Delivering Nucleic Acids to Cells

ABSTRACT

Cyclic amidinium containing compounds and their methods of preparation are described. Compositions containing these compounds facilitate delivery of biologically active polymers to cells in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/154,494,pending, which is a divisional of application Ser. No. 10/445,696, filedMay 27, 2003, now U.S. Pat. No. 7,220,400, which claims the benefit ofU.S. Provisional Applications No. 60/383,201 filed May 24, 2002 and No.60/411,332 filed Sep. 17, 2002.

BACKGROUND OF THE INVENTION

Biologically active polymers such as proteins, enzymes, and nucleicacids (DNA and RNA) have been delivered to the cells using amphipathiccompounds that contain both hydrophobic and hydrophilic domains.Typically these amphipathic compounds are organized into vesicularstructures such as liposomes, micellar, or inverse micellar structures.Liposomes can contain an aqueous volume that is entirely enclosed by amembrane composed of lipid molecules (usually phospholipids) (New 1990).Positively-charged, neutral, and negatively-charged liposomes have beenused to deliver nucleic acids to cells. For example, plasmid DNAexpression in the liver has been achieved via liposomes delivered bytail vein or intraportal routes. Positively-charged micelles have alsobeen used to package nucleic acids into complexes for the delivery ofthe nucleic acid to cells

Polymers have also been widely used for the delivery of biologicallyactive polymers to cells. A number of drug delivery applications utilizepolymer matrices as the drug carrier. Polymers have been used for thedelivery of nucleic acids (polynucleotides, oligonucleotides, and RNA's)to cells for research and therapeutic purposes. This application hasbeen termed transfection and gene therapy or anti-sense therapy,respectively. One of the several methods of nucleic acid delivery to thecells is the use of DNA-polycation complexes. It was shown that cationicproteins like histones and protamines or synthetic polymers likepolylysine, polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine are effective intracellular delivery agents.Polycations are a very convenient linker for associating specificreceptors or ligands with the nucleic acid-polycation complex, and as aresult, nucleic acid-polycation complexes can be targeted to specificcell types. Polycations also protect nucleic acid in the complexesagainst nuclease degradation. This protection is important for bothextracellular and intracellular preservation of nucleic acid.

The main mechanism of nucleic acid translocation to the intracellularspace might be non-specific adsorptive endocytosis. Gene delivery usingcationic polymers may be increased by preventing endosome acidificationsuch as with NH₄Cl or chloroquine. Some polymers, such aspolyethylenimine and poly(propyl acrylic) acid, may also possessmembrane disruptive or endosomalytic properties. Several reports haveattributed the gene delivery properties of polyethylenimine to abuffering or proton sponge effect. Disruption of endosomes has also beenreported as a result of linking to the polycation endosomal-disruptiveagents such as fusion peptides or adenoviruses.

Polycations can also facilitate nucleic acid condensation. The volumewhich one DNA molecule occupies in a complex with polycations isdramatically lower than the volume of a free DNA molecule. The size of aDNA/polymer complex is probably critical for gene delivery in vivo. Interms of intravenous injection, DNA needs to cross the endothelialbarrier and reach the parenchymal cells of interest. The largestendothelia fenestrae (holes in the endothelial barrier) occur in theliver and have an average diameter of 100 nm. The trans-epithelial poresin other organs are much smaller, for example, muscle endothelium can bedescribed as a structure which has a large number of small pores with aradius of 4 nm, and a very low number of large pores with a radius of20-30 nm. The size of the DNA complexes is also important for thecellular uptake process. After binding to the cells the DNA-polycationcomplex are likely taken up by endocytosis. Therefore, DNA complexessmaller than 100 nm are preferred.

SUMMARY OF THE INVENTION

In a preferred embodiment, we describe compositions for delivering abiologically active polymer to a cell comprising: cyclicamidinium-containing compounds. Cyclic amidiniums are groups derivedfrom the intramolecular cyclization of an amine with an amide (resultingin a dehydrating ring formation) on the same polymer. The resulting ringstructure has a formal positive charge, but may occur in a polymer thathas on overall positive, negative, or neutral charge. The ring size maybe 5 to 9 atoms but most favorably a 5, 6 or 8-membered ring, such as animidazolinium, a 1,3-piperazinium ring, 1,3-nitrogen-8-memberedheterocyclic ring. The amide may be alkyl, aryl, or may have anysubstitution or functionality. The polymers and compositions of theinvention, are useful for the delivery of compounds to cells in vitroand in vivo. Compounds that may be delivered to cells may be selectedfrom the list comprising: polynucleotides, oligonucleotides, DNA, RNA,siRNA, DNA and RNA analogs, and biologically active polymers.

The current invention also relates to compositions comprising:polycations derived from the intramolecular cyclization of acylatedlinear or branched polyethylenimine. The polymers obtained arecopolymers of the subunits selected from the group consisting of: ethylamine, 2-substituted imidazolinium, and N-acyl-ethyleneamine. Apreferred polyimidazolinium is derived from N-propionyl linearpolyethylenimine. The polymers and compositions of the invention can beused for the delivery of compounds to cells in vitro and in vivo.Compounds that can be delivered to cells using the described polymersmay be selected from the list comprising: polynucleotides,oligonucleotides, DNA, RNA, siRNA, DNA and RNA analogs, biologicallyactive polymers.

The present invention provides for the transfer of polynucleotides, andother biologically active polymers into cells in vitro and in vivo,comprising: associating the biologically active polymer with a compoundselected from the group consisting of: cyclic amidinium-containingcompounds, poly cyclic amidinium-containing compounds,imidazolinium-containing compounds, polyimidazolinium compounds,imidazoline-containing compounds, polyimidazoline compounds,1,3-piperazinium ring-containing compounds and poly-1,3-piperaziniumring compounds; and delivering the complex to the cell. The complex maybe delivered intravasculary, intrarterially, intravenously, orally,intraduodenaly, via the jejunum (or ileum or colon), rectally,transdermally, subcutaneously, intramuscularly, intraperitoneally,intraparenterally, via direct injections into tissue, via mucosalmembranes, or into ducts of the salivary or other exocrine glands. Thepolymer may be modified to contain one or more functional groups thatenhance delivery of the polynucleotide of other biologically activepolymer to the cell.

Further objects, features, and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B. Illustrations of the chemical structures for (A) cyclicamidine and (B) cyclic amidinium.

FIG. 2. Illustration of a transamidation reaction.

FIG. 3. Illustrations of the chemical structures for imidazoliniumheterocyclic rings.

FIG. 4. Illustration of imidazolinium formation fromN-acyl-N,N′-diphenyl-ethylenediamine

FIG. 5. Illustration of imidazolinium formation from the N-alkylation ofan imidazoline ring.

FIG. 6. Illustration of the formations of a polyimidazoline polymer anda poly-imidazolinium polymer.

FIG. 7. Illustrations for the acid hydrolysis and base hydrolysis of anamide.

FIG. 8. Illustration of the chemical structure for 2-imidazoline.

FIG. 9. Illustration of the chemical structure for 1,3-piperazinium.

FIG. 10. Illustration of the chemical polymerization to form branchedpolyethyleneimine.

FIG. 11. Illustration of the chemical polymerization to form linearpolyethyleneimine.

DETAILED DESCRIPTION OF THE INVENTION

We describe a new class of cyclic amidinium-containing compounds(FIG. 1) that facilitate delivery of biologically active polymers tocells. Specifically, the invention relates compounds containing cationicheterocyclic ring structures derived from the intramolecular1,2-addition of an amine to an amide, followed by a dehydration.Polymers with an amine four to eleven, and more preferably four toseven, bonds away from an amide carbonyl which is able to undergo thisintermolecular cyclization are envisioned in this invention. Thesepolymers contain 1,3-heterocyclic ring systems that possess a formalpositive charge. The amide can be formed on a preformed polymer via anacylation reaction, well know in the art. Additionally, an amidecontaining polymer may be synthesized; for example, the polymerresulting from the polymerization of a 2-oxazoline. A controlled amidehydrolysis can be conducted to synthesize a polymer with the desiredproportion of amide containing units and free amine (or protonated aminesalt) containing units. The resulting polymer can then be bufferedbetween pH 2-8 and left to undergo the cyclization reaction. Thesepolymers may be selected from the list comprising: partially acylatedpolyethyleinimines (lPEI, brPEI), partially acylated polyvinylamine,partially acylated polyallylamine, and partially acylatedpolypropylamine. The ring structures formed may be selected from thelist comprising: imidazolinium rings, 1,3-piperazinium rings, and1,3-nitrogen-8-membered heterocyclic ring.

FIG. 1A depicts an amidine ring. The amidine ring may be alkylated onthe nitrogen to afford an amidinium ring (FIG. 1B). The amidinium ringcan also form from the intramolecular cyclization and dehydration of anamide and an amine.

In an amidinium (amidinium subunit), substituents R1, R2, R3 canindependently be a hydrogen radical or a carbon radical with anysubstitution. The ring can be from 5 to 12 atoms, and can containadditional heteroatoms in addition to the 1,3-Nitrogen atoms.Additionally, the ring can be substituted in other positions,independently being a hydrogen radical, a carbon radical with anysubstitution, or a heteroatom radical with any substitution. Thecounterion (X) can be any counterion. Counterions include, but are notlimited to chloride, bromide, iodide, and tosylate.

A polyamidinium is a polymer (random copolymer, block copolymer, orother copolymer) containing two or more amidinium subunits. Apolyamidinium also means a homopolymer of the amidinium subunit. Theamidinium subunit can be in the main chain of the polymer or as a sidechain off of the polymer main chain. The polymer can be a net neutralpolymer, a polycation, or a polyanion.

A poly-2-amidine is a polymer (random polymer, or block polymer)containing one or more amidine subunits. A poly-2-amidine also means ahomopolymer of the 2-amidine subunit. The amidine subunit can be in themain chain of the polymer or as a side chain off of the polymer mainchain. The polymer can be a net neutral polymer, a polycation, or apolyanion.

In a 2-amidine (amidine subunit), substituents R1, R2 can independentlybe a hydrogen radical or a carbon radical with any substitution. Thering can be from 5 to 12 atoms, and can contain additional heteroatomsin addition to the 1,3-Nitrogen atoms. Additionally, the ring can besubstituted in other positions, independently being a hydrogen radical,a carbon radical with any substitution, or a heteroatom radical with anysubstitution.

Amides undergo a variety of chemical transformations. For example,amines are known to react with amides or lactams (cyclic amides) in atransamidation reaction, resulting in the formation of a new amide bond(and a corresponding ring expansion product in the case of lactams)(Chimishkyan 1985; Garcia 1982; Stach 1988; Krammer 1977; Krammer 1978;Askitoglu 1985). Transamidation reactions are know to be substitutiondependent on both the amine and the amide. FIG. 2

Intramolecular transamidation reactions on both alkyl amides and arylamides have been reported. Intramolecular reactions between amines andamides have also been reported. In a number of studies, a dehydration ofthe initial addition product has been observed. This reaction leads tothe formation of a heterocyclic ring, in particular the formation of animidazolinium ring system (FIG. 3A). (May et al. 1951; Jaenicke et al1959; Hafferl, et al. 1963; Barnett et al. 1966). In particular, thecyclization reaction has been studied on monoacylated derivatives ofN,N′-diphenylethylenediamine (FIG. 4). As the imidazolinium ring (FIG.4) is formed in the cyclization, there is a shift in the UV absorbancefor the material, with a new absorbance growing in at μ=220-240 nmrange. Reports indicate that the reaction is reversible, depending onthe pH of the solution, favoring an imidazolinium under near neutral toacidic conditions, and as a ring opened β-amino amide under basicconditions. In addition to the addition-dehydration preparation methods,an imidazolinium ring can be formed via the N-alkylation of thecorresponding imidazoline (FIG. 5) (Anderson et al. 1986; Gruseck et al.1987; Fernandez et al. 1987; Salerno et al. 1992; Perillo et al. 1975),through a nitrilium ion cyclization (Leonard 1965; Pfeil et al. 1965;Leonard et al. 1967), or via a cyclization reaction of adiazapentadinium salt (Lloyd et al. 1978).

Imidazolinium systems have been developed as surfactants for use ascorrosion inhibitors. Additionally, imidazolinium lipids have beenprepared, and formulated with DNA in a complex (Lasic et al. 1997), andwith DNA for an in vivo liposomal delivery system (Niven et al. 1998).Imidazoline and imidazolinium polymers have also been prepared (FIG. 6).These polymer systems have found use as flocculation aids for thedewatering of sludge. We now show that poly cyclic amidinium systems areuseful for delivery of nucleic acid to cells.

Hydrolysis of amides is a well know method in the art, and can beaccomplished under both acid and base catalyzed procedures (FIG. 7).Both methods result in the formation of 1 equivalent of carboxylic acid(or carboxylate ion) and 1 equivalent of amine (or ammonium ion), andare essentially irreversible. Generally, base catalyzed amid hydrolysisis less reliable than acidic hydrolysis, and is therefore not generallyused. Amide hydrolysis is not as facile as the hydrolysis of esters, andusually requires more stringent conditions, such as elevatedtemperature.

Polyimidazolinium: A polyimidazolinium is a polymer (random copolymer,block copolymer, or other copolymer) containing two or moreimidazolinium subunits. A polyimidazolium also means a homopolymer ofthe imidazolinium subunit. The imidazolinium subunit can be in the mainchain of the polymer or as a side chain off of the polymer main chain.The polymer can be a net neutral polymer, a polycation, or a polyanion.

Imidazolinium Subunit In an imidazolinium (imidazolinium subunit),substituents R1, R2, R3, R4a, R4b, R5a, and R5b (FIG. 3) canindependently be a hydrogen radical or a carbon radical with anysubstitution. The counterion (X) can be any counterion. Counterionsinclude, but are not limited to chloride, bromide, iodide, and tosylate.An imidazolinium ring can be formed via the N-alkylation of thecorresponding imidazoline (FIG. 5).

Poly-2-Imidazoline: A poly-2-imidazoline is a polymer (random polymer,or block polymer) containing two or more imidazoline subunits. Apoly-2-imidazoline also means a homopolymer of the 2-imidazolinesubunit. The imidazoline subunit can be in the main chain of the polymeror as a side chain off of the polymer main chain. The polymer can be anet neutral polymer, a polycation, or a polyanion.

2-Imidazoline Subunit: In a 2-imidazoline (imidazoline subunit),substituents R1, R2, R4a, R4b, R5a, and R5b (FIG. 8) can independentlybe a hydrogen radical or a carbon radical with any substitution.

Poly-1,3-piperazinium: A poly-1,3-piperazinium is a polymer (randomcopolymer, block copolymer, or other copolymer) containing two or more1,3-piperazinium subunits. A poly 1,3-piperazinium also means ahomopolymer of the 1,3-piperazinium subunit. The 1,3-piperaziniumsubunit can be in the main chain of the polymer or as a side chain offof the polymer main chain. The polymer can be a net neutral polymer, apolycation, or a polyanion.

1,3-piperazinium (1,3-piperazinium Subunit): (FIG. 9) In an1,3-piperazinium (1,3-piperazinium Subunit), substituents R1, R2, R3,R4a, R4b, R5a, R5b, R6a, and R6b can independently be a hydrogen radicalor a carbon radical with any substitution. The counterion (X) can be anycounterion. Counterions include, but are not limited to chloride,bromide, iodide, and tosylate.

Poly cyclic amidinium polymers can be prepared from a class of polymerscalled polyethylenimines. Polyethylenimines themselves have frequentlybeen used in the area of nucleic acid delivery. Polyethylenimine isknown in two versions prepared via two distinctive polymerizationprocesses. Branched polyethyleneimine (brPEI) is prepared through thepolymerization of aziridine (ethyleneimine) (FIG. 10). During thepolymerization reaction, the primary and secondary amines formed can actas nucleophiles, attacking another aziridinium molecule to produce to ahighly branched structure. The composition of commercially availablebrPEI has recently been evaluated to contain an amino group ratio ofprimary amine:secondary amine:tertiary amine of 1:1:1 (Kissel 2000).BrPEI can also be polymerized under conditions to obtain a lower degreeof branching, resulting in an amino group ratio of primaryamine:secondary amine:tertiary amine of 1:2:1.

Linear polyethyleneimine (lPEI) has been shown to exhibit lowercytotoxicity as compared with brPEI, and is an effective gene transferagent. (Boussif et al. 1995; Behr 1999; Behr 1999; Reny 1998, U.S. Pat.No. 6,013,240; Park et al 2001). Linear PEI is obtained via aring-opening polymerization of N-alkyl-2-oxazoline, followed by theacid-induced deprotection of the resulting amide (FIG. 11). The polymeris represented by the general formula:HO—(CH₂)₂N—(CH₂—CH₂—NH)_(n)—(CH₂)₂—N—R, wherein the R group is derivedfrom the initiator used in the polymerization reaction (the figuredepicts the final oxazolinium to be hydrolysed to the terminalhydroxyl). For example, if the polymerization is initiated with methyliodide, the R group in the structure would be CH₃. Linear PEI sold forgene therapy applications (Polyplus-transfection SAS, JetPEI; Fermentas;ExGen500), has been described as a linear polymer of ethylamine,containing all secondary amines, as depicted in figure.

Polyethyleneimines can be acylated on the nitrogen atom according towell know methods in the art. The resulting polymers have an amidecarbonyl four bonds from an amine. These polymers can be taken up inaqueous solutions at or below pH 8, and gently warmed or stored forperiods at RT to facilitate the cyclization reaction to form the cyclicamidinium ring, in particular a imidazolinium ring. The resultingpolymer is therefore a combination of ethylenimine, N-acyl ethylenimine(from amides that did not cyclize), and 2-substituted imidazoliniummonomers. The proportion of the monomers in the final polymer can becontrolled by the level of acylation of the polyethylenimine.Additionally, it is envisioned that additional functionality can beeasily included in the system by attaching the functionality to anitrogen atom on the polyethylenimine via an acylation. The resultingamide would be available to undergo the cyclization reaction, affordinga 2-substituted imidazolinium that contains the functionality tetheredto the 2 position of the imidazolinium ring.

The size of a nucleic acid/polycation complex may be a factor for genedelivery to cells, particularly in vivo. Often, the size of a nucleicacid of interest is large, too large to facilitate delivery withoutcompacting, or condensing, the nucleic acid. For in vivo delivery, thecomplex needs to cross the endothelial barrier to reach parenchymalcells. The largest endothelial fenestrae (holes in the endothelialbarrier) occur in the liver and have average diameter of 100 nm. Thetrans-epithelial pores in other organs are much smaller. Muscleendothelium, for example, can be described as a structure which has alarge number of small pores, of radius 4 nm, and a very low number oflarge pores with a radius of 20-30 nm. The size of the complex may alsobe important for cellular uptake, with smaller particles being morereadily endocytosed.

There are two major approaches for compacting nucleic acid: 1.Multivalent cations with a charge of three or higher have been shown tospontaneously condense nucleic acid under appropriate buffer conditions.These multivalent cations include spermidine, spermine, Co(NH₃)₆³⁺,Fe³⁺, and natural or synthetic polymers such as histone H1,protamine, polylysine, and polyethylenimine. One analysis has shownnucleic acid condensation to be favored when 90% or more of the chargesalong the sugar-phosphate backbone are neutralized (Wilson et al. 1979).2. Polymers (neutral or anionic) can increase repulsion between nucleicacid and its surroundings, and have been shown to compact nucleic acid.Most significantly, spontaneous nucleic acid self-assembly andaggregation process have been shown to result from the confinement oflarge amounts of nucleic acid, due to excluded volume effect (Strzelecka1990a; Strzelecka 1990b). Since self-assembly is associated with locallyor macroscopically crowded nucleic acid solutions, it is expected thatnucleic acid insertion into small water cavities with a size comparableto the nucleic acid will tend to form mono or oligomolecular compactstructures.

The mechanism of nucleic acid condensation is not clear. Theelectrostatic force between unperturbed helices arises primarily from acounterion fluctuation mechanism requiring multivalent cations and playsa major role in nucleic acid condensation. The hydration forcespredominate over electrostatic forces when the nucleic acid helicesapproach closer then a few water diameters. In a case of nucleicacid/polymeric polycation interactions, nucleic acid condensation is amore complicated process than the case of low molecular weightpolycations. Different polycationic proteins can generate toroid and rodformations with nucleic acid at a positive to negative charge ratio of2-5. T4 DNA complexes with polyarginine or histone can form two types ofstructures; an elongated structure with a long axis length of about 350nm (like free DNA) and dense spherical particles. Both forms existsimultaneously in the same solution. The reason for the co-existence ofthe two forms can be explained as an uneven distribution of thepolycation chains among the nucleic acid molecules. The unevendistribution generates two thermodynamically favorable conformations.

The electrophoretic mobility of nucleic acid/polycation complexes canchange from negative to positive in the presence of excess polycation.It is likely that large polycations don't completely align along nucleicacid but form polymer loops that interact with other nucleic acidmolecules. The rapid aggregation and strong intermolecular forcesbetween different nucleic acid molecules may prevent the slow adjustmentbetween helices needed to form tightly packed orderly particles.

Poly cyclic amidinium polymers may be prepared such that they havesufficient positive charge to form complexes with nucleic acid.Alternatively, cyclic amidinium monomers and small poly cyclic amidiniummonomers may by polymerized using a process termed templatepolymerization. Low molecular weight cations with valency, i.e. charge,<+3 fail to condense DNA in aqueous solutions under normal conditions.However, cationic molecules with the charge <+3 can be polymerized inthe presence of polynucleic acid and the resulting polymers can causethe polynucleic acid to condense into compact structures. Such anapproach is known in synthetic polymer chemistry as templatepolymerization. During this process, monomers (which are initiallyweakly associated with the template) are positioned along a template'sbackbone, thereby promoting their polymerization. Weak electrostaticassociation of the nascent polymer and the template becomes strongerwith chain growth of the polymer. Trubetskoy et al used two types ofpolymerization reactions to achieve DNA condensation: steppolymerization and chain polymerization (Trubetskoy et al. 1998; U.S.Ser. No. 08/778,657; U.S. Ser. No. 09/000,692; U.S. Ser. No. 97/240,89;U.S. Ser. No. 09/070,299; U.S. Ser. No. 09/464,871).Bis(2-aminoethyl)-1,3-propanediamine (AEPD), a tetramine with 2.5positive charges per molecule at pH 8 was polymerized in the presence ofplasmid DNA using cleavable disulfide amino-reactive cross-linkersdithiobis (succinimidyl propionate) anddimethyl-3,3′-dithiobispropionimidate. Both reactions yieldedDNA/polymer complexes with significant retardation in agaroseelectrophoresis gels, thus demonstrating significant binding and DNAcondensation. Blessing et al used a bisthiol derivative of spermine andreaction of thiol-disulfide exchange to promote chain growth. Thepresence of DNA accelerated the polymerization reaction as measured bythe rate of disappearance of free thiols in the reaction mixture(Blessing et al. 1998).

In order to increase the stability of a poly(cyclic amidinium)/nucleicacid complex, the polymer may be crosslinked. The stability of nucleicacid particles, based on polynucleic acid condensation, is generally lowin vivo or in the presence of other polyanions because the complexeseasily engage in polyanion exchange reactions. The process of exchangeconsists of two stages: 1) rapid formation of a triple nucleicacid-polycation-polyanion complex, 2) slow substitution of one polyanion(nucleic acid) with another. At equilibrium conditions, this processresults in formation of a new binary complex and an excess of a thirdpolyanion. The presence of low molecular weight salt can greatlyaccelerate such exchange reactions, which often result in completedisassembly of condensed nucleic acid particles. Hence, it is desirableto obtain more colloidally stable structures in which the nucleic acidis retained in condensed form in complex with the polycation independentof environment conditions.

In typical nucleic acid/polycation complexes, in which the nucleic acidis completely condensed, unpaired positive charges on the polycationremain available. If the polycation contains reactive groups, such asprimary amines, these unpaired positive charges may be modified. Thismodification allows practically limitless possibilities of modulatingcolloidal properties of the complexes via chemical modifications of thecomplex. We have demonstrated the utility of such reactions using aDNA/poly-L-lysine (DNA/PLL) system crosslinked with the bifunctionalcross-linking reagent dimethyl-3,3′-dithiobispropionimidate (DTBP) whichreacts with primary amino groups (Trubetskoy et al. 1999b; U.S. Ser. No.08/778,657; U.S. Ser. No. 09/000,692; U.S. Ser. No. 97/24,089; U.S. Ser.No. 09/070,299; U.S. Ser. No. 09/464,871). Similar results were achievedwith other polycations including poly(allylamine) and histone H1. Theuse of another bifunctional reagent, glutaraldehyde, has been describedfor stabilization of DNA complexes with cationic peptide CWK18 (Adam etal. 1999).

The caging approach described above could lead to more colloidallystable condensed nucleic acid-containing particles. However, for somedelivery applications it may be desirable to alter the surface charge ofa nucleic acid/polycation complex. The phenomenon of surface rechargingis well known in colloid chemistry and is described in great detail forlyophobic/lyophilic systems (for example, silver halide hydrosols).Addition of polyion to a suspension of latex particles withoppositely-charged surface leads to the permanent absorption of thispolyion on the surface and, upon reaching appropriate stoichiometry,reversing the surface charge. We have demonstrated that similar layeringof polyelectrolytes can be achieved on the surface of DNA/polycationparticles (Trubetskoy et al. 1999b). The principal DNA-polycation(DNA/pC) complex used in this study was DNA/PLL (1:3 charge ratio)formed in low salt 25 mM HEPES buffer and recharged with increasingamounts of various polyanions. The DNA particles were characterizedafter addition of a third polyion component to a DNA/polycation complex(Trubetskoy et al. 1999c). It was found that certain polyanions, such aspoly(methacrylic acid) and poly(aspartic acid), decondensed DNA inDNA/PLL complexes. Polyanions of lower charge density, such assuccinylated PLL and poly(glutamic acid), did not decondense DNA inDNA/PLL (1:3) complexes even when added in 20-fold charge excess to PLL.Further studies have found that displacement effects are salt-dependent.Measurement of ζ-potential of DNA/PLL particles during titration withSPLL revealed the change of particle surface charge at approximately thecharge equivalency point. Thus, it can be concluded that addition of lowcharge density polyanion to the cationic DNA/PLL particles results inparticle surface charge reversal while maintaining condensed DNA coreintact. Finally, DNA/polycation complexes can be both recharged andcrosslinked or caged (U.S. Ser. No. 08/778,657, U.S. Ser. No.09/000,692, U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070,299, and U.S.Ser. No. 09/464,871). This recharging polyanion layer can be crosslinkedto itself or to the polycations in the complex to increase colloidalstability.

Several modifications of nucleic acid/cation particles have been createdto circumvent the nonspecific interactions of nucleic acid-cationparticles and the toxicity of cationic particles. Examples of thesemodifications include attachment of steric stabilizers, e.g.polyethylene glycol, which inhibit nonspecific interactions between thecation and biological polyanions.

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers, which have two to about 80 monomers, andpolymers having more than 80 monomers. The polymer can be linear,branched network, star, comb, or ladder types of polymer. The polymercan be a homopolymer in which a single monomer is used or can becopolymer in which two or more monomers are used. Types of copolymersinclude alternating, random, block and graft.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer. “Most step-reactionpolymerizations are condensation processes and most chain-reactionpolymerizations are addition processes” (Stevens 1990). Templatepolymerization can be used to form polymers from daughter polymers.

Chain Polymerization In chain-reaction polymerization growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain terminating step. Thepolymerization rate remains constant until the monomer is depleted.Monomers containing (but not limited to) vinyl, acrylate, methacrylate,acrylamide, methacrylamide groups can undergo chain reaction which canbe radical, anionic, or cationic. Chain polymerization can also beaccomplished by cycle or ring opening polymerization. Several differenttypes of free radical initiators could be used that include peroxides,hydroxy peroxides, and azo compounds such as2,2′-Azobis(-amidinopropane) dihydrochloride (AAP).

Types of Monomers

A wide variety of monomers can be used in the polymerization processes.These include positive charged organic monomers such as amine salts,imidine, guanidine, imine, hydroxylamine, hydrozyine, heterocycle(salts) like imidazole, pyridine, morpholine, pyrimidine, or pyrene. Theamines could be pH-sensitive in that the pKa of the amine is within thephysiologic range of 4 to 8. Specific amines include spermine,spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties; carbohydrates,polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobicgroups.

Monomers can also be intercalating agents such as acridine, thiazoleorange, or ethidium bromide. Monomers can also contain chemical moietiesthat can be modified before or after the polymerization including (butnot limited to) amines (primary, secondary, and tertiary), amides,carboxylic acid, ester, hydroxyl, hydrazine, alkyl halide, aldehyde, andketone.

A polycation is a polymer containing a net positive charge. Thepolycation can contain monomer units that are charge positive, chargeneutral, or charge negative, however, the net charge of the polymer mustbe positive. A polycation also can mean a non-polymeric molecule thatcontains two or more positive charges. A polyanion is a polymercontaining a net negative charge, for example polyglutamic acid. Thepolyanion can contain monomer units that are charge negative, chargeneutral, or charge positive, however, the net charge on the polymer mustbe negative. A polyanion can also mean a non-polymeric molecule thatcontains two or more negative charges. The term polyion includespolycation, polyanion, zwitterionic polymers, and neutral polymers. Theterm zwitterionic refers to the product (salt) of the reaction betweenan acidic group and a basic group that are part of the same molecule.

The polymers have other functional groups that increase their utility.These groups can be incorporated into monomers prior to polymerformation or attached to the polymer after its formation.

Functional group. Functional groups include cell targeting signals,nuclear localization signals, compounds that enhance release of contentsfrom endosomes or other intracellular vesicles (releasing signals), andother compounds that alter the behavior or interactions of the compoundor complex to which they are attached.

Cell targeting signals are any signals that enhance the association ofthe biologically active polymer with a cell. These signals can modify abiologically active polymer such as drug or nucleic acid and can directit to a cell location (such as tissue) or location in a cell (such asthe nucleus) either in culture or in a whole organism. The signal mayincrease binding of the compound to the cell surface and/or itsassociation with an intracellular compartment. By modifying the cellularor tissue location of the foreign gene, the function of the biologicallyactive polymer can be enhanced. The cell targeting signal can be, but isnot limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate,(non-expressing) polynucleic acid or synthetic compound. Cell targetingsignals such as ligands enhance cellular binding to receptors. A varietyof ligands have been used to target drugs and genes to cells and tospecific cellular receptors. The ligand may seek a target within thecell membrane, on the cell membrane or near a cell. Binding of ligandsto receptors typically initiates endocytosis. Ligands include agentsthat target to the asialoglycoprotein receptor by usingasialoglycoprotein or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with thiol, sulfhydryl, or disulfide groups on cellscan also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as lipids, fatty acids,cholesterol, dansyl compounds, and amphotericin derivatives. In additionviral proteins could be used to bind cells.

After interaction of a compound or complex with the cell, othertargeting groups can be used to increase the delivery of thebiologically active polymer to certain parts of the cell.

Nuclear localizing signals enhance the targeting of the pharmaceuticalinto proximity of the nucleus and/or its entry into the nucleus duringinterphase of the cell cycle. Such nuclear transport signals can be aprotein or a peptide such as the SV40 large T antigen NLS or thenucleoplasmin NLS. These nuclear localizing signals interact with avariety of nuclear transport factors such as the NLS receptor(karyopherin alpha) which then interacts with karyopherin beta. Thenuclear transport proteins themselves could also function as NLS's sincethey are targeted to the nuclear pore and nucleus. For example,karyopherin beta itself could target the DNA to the nuclear porecomplex. Several peptides have been derived from the SV40 T antigen.Other NLS peptides have been derived from the hnRNP A1 protein,nucleoplasmin, c-myc, etc.

Many biologically active polymers, in particular large and/or chargedcompounds, are incapable of crossing biological membranes. In order forthese compounds to enter cells, the cells must either take them up byendocytosis, i.e., into endosomes, or there must be a disruption of thecellular membrane to allow the compound to cross. In the case ofendosomal entry, the endosomal membrane must be disrupted to allow formovement out of the endosome and into the cytoplasm. Either entrypathway into the cell requires a disruption or alteration of thecellular membrane. Compounds that disrupt membranes or promote membranefusion are called membrane active compounds. These membrane activecompounds, or releasing signals, enhance release of endocytosed materialfrom intracellular compartments such as endosomes (early and late),lysosomes, phagosomes, vesicle, endoplasmic reticulum, Golgi apparatus,trans Golgi network (TGN), and sarcoplasmic reticulum. Release includesmovement out of an intracellular compartment into the cytoplasm or intoan organelle such as the nucleus. Releasing signals include chemicalssuch as chloroquine, bafilomycin or Brefeldin A1 and the ER-retainingsignal (KDEL sequence), viral components such as influenza virushemagglutinin subunit HA-2 peptides and other types of amphipathicpeptides. The control of when and where the membrane active compound isactive is crucial to effective transport. If the membrane active agentis operative in a certain time and place it would facilitate thetransport of the biologically active polymer across the biologicalmembrane. If the membrane active compound is too active or active at thewrong time, then no transport occurs or transport is associated withcell rupture and cell death. Nature has evolved various strategies toallow for membrane transport of biologically active polymers includingmembrane fusion and the use of membrane active compounds whose activityis modulated such that activity assists transport without toxicity. Manylipid-based transport formulations rely on membrane fusion and somemembrane active peptides' activities are modulated by pH. In particular,viral coat proteins are often pH-sensitive, inactive at neutral or basicpH and active under the acidic conditions found in the endosome.

Another functional group comprises compounds, such as polyethyleneglycol, that decrease interactions between molecules and themselves andwith other molecules. Such groups are useful in limiting interactionssuch as between serum factors and the molecule or complex to bedelivered.

By delivered we mean that the polynucleotide or other biologicallyactive polymer becomes associated with the cell. The polynucleotide orother biologically active polymer can be on the membrane of the cell orinside the cytoplasm, nucleus, or other organelle of the cell. Theprocess of delivering a nucleic acid to a cell has been commonly termedtransfection or the process of transfecting and also it has been termedtransformation. The term transfecting as used herein refers to theintroduction of foreign nucleic acid or other biologically activepolymer into cells. The biologically active polymer could be used forresearch purposes or to produce a change in a cell that can betherapeutic. The delivery of nucleic acid for therapeutic purposes iscommonly called gene therapy. The delivery of nucleic acid can lead tomodification of the genetic material present in the target cell. Theterm stable transfection or stably transfected generally refers to theintroduction and integration of exogenous nucleic acid into the genomeof the transfected cell. The term stable transfectant refers to a cellwhich has stably integrated foreign nucleic acid into the genomic DNA.Stable transfection can also be obtained by using episomal vectors thatare replicated during the eukaryotic cell division (e.g., plasmid DNAvectors containing a papilloma virus origin of replication, artificialchromosomes). The term transient transfection or transiently transfectedrefers to the introduction of foreign nucleic acid into a cell where theforeign nucleic acid does not integrate into the genome of thetransfected cell. A biologically active polymer is a compound having thepotential to react with biological components. Pharmaceuticals,proteins, peptides, hormones, cytokines, antigens and nucleic acids areexamples of biologically active polymers. These processes can be usedfor transferring nucleic acids or biomolecules into cells or an organismsuch as for drug delivery, or may also be used for analytical methods.

A delivery system is the means by which a biologically active polymerbecomes delivered. That is all compounds, including the biologicallyactive polymer itself, that are required for delivery and all proceduresrequired for delivery including the form (such volume and phase (solid,liquid, or gas)) and method of administration (such as but not limitedto oral or subcutaneous methods of delivery).

The term polynucleotide, or nucleic acid or polynucleic acid, is a termof art that refers to a polymer containing at least two nucleotides.Nucleotides are the monomeric units of polynucleotide polymers.Polynucleotides with less than 120 monomeric units are often calledoligonucleotides. Natural nucleic acids have a deoxyribose- orribose-phosphate backbone. An artificial or synthetic polynucleotide isany polynucleotide that is polymerized in vitro or in a cell free systemand contains the same or similar bases but may contain a backbone of atype other than the natural ribose-phosphate backbone. These backbonesinclude: PNAs (peptide nucleic acids), phosphorothioates,phosphorodiamidates, morpholinos, and other variants of the phosphatebackbone of native nucleic acids. Bases include purines and pyrimidines,which further include the natural compounds adenine, thymine, guanine,cytosine, uracil, inosine, and natural analogs. Synthetic derivatives ofpurines and pyrimidines include, but are not limited to, modificationswhich place new reactive groups such as, but not limited to, amines,alcohols, thiols, carboxylates, and alkylhalides. The term baseencompasses any of the known base analogs of DNA and RNA including, butnot limited to, 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, β-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. The term polynucleotide includes deoxyribonucleicacid (DNA) and ribonucleic acid (RNA) and combinations on DNA, RNA andother natural and synthetic nucleotides.

DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, partsof a plasmid DNA, genetic material derived from a virus, linear DNA,vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, recombinant DNA, chromosomal DNA, anoligonucleotide, anti-sense DNA, or derivatives of these groups. RNA maybe in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (smallnuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), in vitropolymerized RNA, recombinant RNA, chimeric sequences, anti-sense RNA,siRNA (small interfering RNA), ribozymes, or derivatives of thesegroups. An anti-sense polynucleotide is a polynucleotide that interfereswith the function of DNA and/or RNA. Antisense polynucleotides include,but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA,RNA and the like. SiRNA comprises a double stranded structure typicallycontaining 15-50 base pairs and preferably 21-25 base pairs and having anucleotide sequence identical or nearly identical to an expressed targetgene or RNA within the cell. Interference may result in suppression ofexpression. The polynucleotide can be a sequence whose presence orexpression in a cell alters the expression or function of cellular genesor RNA. In addition, DNA and RNA may be single, double, triple, orquadruple stranded. Double, triple, and quadruple strandedpolynucleotide may contain both RNA and DNA or other combinations ofnatural and/or synthetic nucleic acids.

A delivered polynucleotide can stay within the cytoplasm or nucleusapart from the endogenous genetic material. Alternatively, DNA canrecombine with (become a part of) the endogenous genetic material.Recombination can cause DNA to be inserted into chromosomal DNA byeither homologous or non-homologous recombination.

A polynucleotide can be delivered to a cell to express an exogenousnucleotide sequence, to inhibit, eliminate, augment, or alter expressionof an endogenous nucleotide sequence, or to affect a specificphysiological characteristic not naturally associated with the cell.Polynucleotides may contain an expression cassette coded to express awhole or partial protein, or RNA. An expression cassette refers to anatural or recombinantly produced polynucleotide that is capable ofexpressing a gene(s). The term recombinant as used herein refers to apolynucleotide molecule that is comprised of segments of polynucleotidejoined together by means of molecular biological techniques. Thecassette contains the coding region of the gene of interest along withany other sequences that affect expression of the gene. A DNA expressioncassette typically includes a promoter (allowing transcriptioninitiation), and a sequence encoding one or more proteins. Optionally,the expression cassette may include, but is not limited to,transcriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include, but is notlimited to, translation termination signals, a polyadenosine sequence,internal ribosome entry sites (IRES), and non-coding sequences.

The polynucleotide may contain sequences that do not serve a specificfunction in the target cell but are used in the generation of thepolynucleotide. Such sequences include, but are not limited to,sequences required for replication or selection of the polynucleotide ina host organism.

A polynucleotide can be used to modify the genomic or extrachromosomalDNA sequences. This can be achieved by delivering a polynucleotide thatis expressed. Alternatively, the polynucleotide can effect a change inthe DNA or RNA sequence of the target cell. This can be achieved byhybridization, multistrand polynucleotide formation, homologousrecombination, gene conversion, or other yet to be described mechanisms.

The term gene generally refers to a polynucleotide sequence thatcomprises coding sequences necessary for the production of a therapeuticpolynucleotide (e.g., ribozyme) or a polypeptide or precursor. Thepolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction) of the full-length polypeptide or fragment are retained.The term also encompasses the coding region of a gene and the includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. The sequencesthat are located 5′ of the coding region and which are present on themRNA are referred to as 5′ untranslated sequences. The sequences thatare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ untranslated sequences. The term geneencompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed introns, intervening regions or intervening sequences.Introns are segments of a gene which are transcribed into nuclear RNA.Introns may contain regulatory elements such as enhancers. Introns areremoved or spliced out from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide. The term non-coding sequences alsorefers to other regions of a genomic form of a gene including, but notlimited to, promoters, enhancers, transcription factor binding sites,polyadenylation signals, internal ribosome entry sites, silencers,insulating sequences, matrix attachment regions. These sequences may bepresent close to the coding region of the gene (within 10,000nucleotide) or at distant sites (more than 10,000 nucleotides). Thesenon-coding sequences influence the level or rate of transcription andtranslation of the gene. Covalent modification of a gene may influencethe rate of transcription (e.g., methylation of genomic DNA), thestability of mRNA (e.g., length of the 3′ polyadenosine tail), rate oftranslation (e.g., 5′ cap), nucleic acid repair, and immunogenicity. Oneexample of covalent modification of nucleic acid involves the action ofLabelIT reagents (Mirus Corporation, Madison, Wis.).

As used herein, the term gene expression refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through transcription of a deoxyribonucleic gene(e.g., via the enzymatic action of an RNA polymerase), and for proteinencoding genes, into protein through translation of mRNA. Geneexpression can be regulated at many stages in the process. Up-regulationor activation refers to regulation that increases the production of geneexpression products (i.e., RNA or protein), while down-regulation orrepression refers to regulation that decrease production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called activators and repressors,respectively.

A biologically active polymer is a compound having the potential toreact with biological components. More particularly, biologically activepolymers utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically active polymer.Biologically active polymers may be selected from the group comprising:pharmaceuticals, proteins, peptides, polypeptides, hormones, cytokines,antigens, viruses, oligonucleotides, and nucleic acids orpolynucleotides.

EXAMPLES Example 1

Formation of an Imidazolinium Containing Copolymer. Linearpolyethylenimine with N-propionyl substitution (10% of the aminesacylated, 90% ethylamine by 1H NMR, Polysciences, Inc., 1H NMR (D₂O,3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as an internalreference) δ3.65-3.45, m, 6H, δ3.15-2.80, m, 47H, δ2.46, q, 2H, δ1.09,t, 3H) was dissolved in water to a concentration of 20 mg/mL. The pH ofthe solution was adjusted to pH 5.0 with hydrochloric acid (12 M). Thesolution was split into two portions. One portion was placed at 4° C.for 48 h. The second portion was placed at 37° C. for 48 h. The sizeexclusion profile for each sample was obtained (250 μg in 250 μL HepesBuffered Saline, BioCad-Sprint, Perseptive Biosystems, Inc., EichromeTechnologies Columns, SPCCS201-30 and SPCCS203-30 connected in series,0.2 M NaCl eluent, monitored at λ210 and λ240). The traces for thesamples were essentially identical, indicating that the molecular weightof the polymer was not effected by the process. The only differencenoted in the example was that the 37° C. sample had a much higherabsorbance at λ240. The UV spectra for each sample was run (0.2 mg/mL inH₂O, λ190 nm-300 nm). The sample stored at 4° C. exhibited a λmax at 205nm, which was identical to the reaction starting material (startingmaterial not shown). The sample kept at 37° C. exhibited a λmax at 235nm. The IR spectra for each sample showed very similar signals, withslight differences in the 1240-1300 cm⁻¹ region and in the 1550-1640cm⁻¹ region. 1H NMR analysis indicated (D₂O,3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as an internalreference) δ3.91, s, 8.2H, δ3.65-3.45, m, 15.6H, δ3.15-2.80, m, 131H,δ2.66, q, 4.4H, δ2.46, q, 2H, δ1.22, t, 7.6H, δ1.09, t, 3H. The newsignals at δ3.91, δ2.66, and δ1.22 are consistent for the imidazoliniumring system. The 1H NMR supports the assignment of 3.3% of the aminesacylated, 7.26% imidazolinium, 89.1% ethylamine. These results show thatthe original ethylamine sections remain and that the new ring systemarises with a corresponding decrease in the amide.

Example 2

In Vivo Mouse Injections of Polyimidazolinium Formulations for LungTransfection. The same samples generated in Example 1 were formulatedinto DNA-containing preparations for intravenous systemic gene transfer.Two complexes were formed.

Complex I. pDNA (50 μg, 25 μL of a 2 μg/μL solution in water) wasdiluted with 10 mM HEPES, 0.29 M glucose, pH 7.5 (200 μL). To thissolution was added the 4° C. sample (400 μg, 20 μL of a 20 mg/mLsolution in water). To this solution was added polyacrylic acid (50 μg,5 μL of a 10 mg/mL solution in water).Complex II. pDNA (50 μg, 25 μL of a 2 μg/μL solution in water) wasdiluted with 10 mM HEPES, 0.29 M glucose, pH 7.5 (200 μL). To thissolution was added the 37° C. sample (400 μg, 20 μL of a 20 mg/mLsolution in water. To this solution was added polyacrylic acid (50 μg, 5μL of a 10 mg/mL solution in water).

Tail vein injections of 250 μL of the complex were preformed on ICR mice(Complex I, n=4; Complex II, n=3) using a 30 gauge, 0.5 inch needle. 30min post injection, the animals were injected with polyacrylic acid(1500 μg, 150 μL of a 10 mg/mL solution in water). One day afterinjection, the animals were sacrificed, and luciferase assays wereconducted on the lung samples. Luciferase expression was determined aspreviously reported (Wolff et al. 1990). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The results indicate thatthe polyimidazolinium complex is more efficient at gene transfer to lungthan the linear polyethylenimine with 10% N-propionyl substitution.

Example 3

pH Effect on the Formation of an Imidazolinium Containing Copolymer.Linear polyethylenimine with N-propionyl substitution (10% of the aminesacylated, 90% ethylamine by 1H NMR, Polysciences, Inc.) was dissolved inwater to a concentration of 20 mg/mL, and the pH was adjusted to 7.4.From this stock, two samples were prepared, one in which the pH wasraised to 8, and a second sample with a pH of 5. The samples were heatedto 37° C. for 64 h and analyzed by UV spectroscopy. The UV spectra foreach sample was run (0.2 mg/mL in H₂O, λ190 nm-300 nm). The samplestored pH 5 showed the largest increase in absorbance at λ235-240. ThepH 8 sample also showed an increase in absorbance at λ235-240,indicating that the cyclization takes place above pH 8.

Example 4

Alkylation of N,N′-Dimethylethylenediamine with Propionyl Chloride.Synthesis of MC1016 (monoaddition) and MC1017 (diaddition).N,N′-Dimethylethylenediamine (500 mg, 11.3 mmol amine, Aldrich ChemicalCompany) was transferred to a flame dried round bottom flask and takenup with dichloromethane (100 mL, 0.1 M). To the resulting solution wasadded diisopropylethylamine (0.99 mL, 730 mg, 5.67 mmol, AldrichChemical Company) and the solution was stirred at RT under N₂. Thereaction mixture was cooled in a dry ice/acetone bath. Propionylchloride (0.49 mL, 525 mg, 5.67 mmol, Aldrich Chemical Company) wasadded dropwise over 5 min to the reaction solution with stirring. Thereaction was allowed to warm to RT with stirring, under N₂. After 30 minat RT, the reaction mixture was analyzed by TLC and mass spectroscopy(Sciex API 150EX) to verify that the reaction was complete. The mixturewas concentrated under reduced pressure, and a portion (100 mg) wasbrought up in H₂O. This solution was purified by HPLC (Aquasil C18column, 0.1% TFA/H₂O and 0.1% TFA/MeCN eluent with a 10-90% (organic)gradient over 20 min, elution rate of 1 mL/min, fractions were collectedat 210 nm). Fractions 1-6 (from two runs) were concentrated underreduced pressure, and lyophilized to afford monopropionylatedN′N-dimethylethylenediamine as a white solid (MC1016, 91.7 mg), asanalyzed by mass spectroscopy (Sciex API 150EX). Fraction 10 (from tworuns) was concentrated under reduced pressure, and lyophilized to affordN,N′-dipropionylated N,N′-dimethylethylene-diamine as a white solid(MC1017, 7.8 mg), analyzed by mass spectroscopy (Sciex API 150EX).

Example 5

Synthesis of 1,3-Dimethy, 2-Ethyl Imidazolinium Chloride.Monopropionylated N′N-dimethylethylenediamine (MC1016) was taken up inwater to a final concentration of 20 mg/mL. The pH of the solution wasadjusted to pH 5 with concentrated HCl. The resulting solution washeated at 70° C. for 72 h. The sample was analyzed by UV Spectroscopy.Analysis indicated an increase in the absorption at λ210 nm for theheated solution relative to a sample stored at 4° C.

Example 6

Synthesis of and Amidinium formation from MonoacylatedPentaethylenehexamine. Pentaethylenehexamine (10 mg, 0.043 mmol, AldrichChemical Company) was taken up in dichloromethane (1 mL). The solutionwas cooled in an ice bath under nitrogen, and acetyl chloride (3.4 mg,0.043 mmol, Aldrich) was added. The solution was allowed to warm toambient temperature and concentrated under reduced pressure. Theresulting oil was taken up in water at a concentration of 20 mg/mL, andthe pH was adjusted to 7 with HCl (6 M). The sample was divided into twoportions. Sample 1 was left at RT while sample 2 was heated at 80° C.After 48 h, the samples were analyzed by UV spectroscopy (Beckman DU530Spectrophotometer). The results indicate an increased absorbance at230-235 for both the RT and the 80° C. samples.

Example 7

Mouse In Vivo Injections of Polyimidazolinium Formulations for LungTransfection. Three samples of polyimidazolinium were prepared frompoly(2-ethyl-2-oxazoline) that was 90% deamidated (PolySciences), bydissolving the polymer at 20 mg/mL in water, adjusting the to pH 7.4,and carrying out cyclization by incubation at elevated temperature.Polyimidazolinium 1 was prepared 6 weeks before use, and was allowed tosit at RT. Polyimidazolinium 2 was prepared 4 weeks before use, and wasallowed to sit at RT. Polyimidazolinium 3 was prepared immediately priorto use. The three polyimidazolinium preparations were analyzed by UVspectroscopy (λ190 nm-300 nm). Polyimidazolinium 1 showed a largeincrease in absorbance at λ235, whereas for Polyimidazolinium 2, thispeak was present, but at a lower amount. Polyimidazolinium did not showa λ235 absorbance. The three polyimidazolinium preparations were used tomake several complexes.

Complex I. pDNA (150 μg, 75 μL of a 2 μg/λL solution in water) wasdiluted with 10 mM HEPES, 0.29 M glucose, pH 7.5 (600 μL). To thissolution was added polyimidazolinium 1 (1200 μg, 60 μL of a 20 mg/mLsolution in water. To this solution was added polyacrylic acid (150 μg,15 μL of a 10 mg/mL solution in water).Complex II. pDNA (150 μg, 75 μL of a 2 μg/λL solution in water) wasdiluted with 10 mM HEPES, 0.29 M glucose, pH 7.5 (600 μL). To thissolution was added polyimidazolinium 2 (1200 μg, 60 μL of a 20 mg/mLsolution in water. To this solution was added polyacrylic acid (150 μg,15 μL of a 10 mg/mL solution in water).Complex III. pDNA (150 μg, 75 μL of a 2 μg/μL solution in water) wasdiluted with 10 mM HEPES, 0.29 M glucose, pH 7.5 (600 μL). To thissolution was added polyimidazolinium 3 (1200 μg, 60 μL of a 20 mg/mLsolution in water. To this solution was added polyacrylic acid (150 μg,15 μL of a 10 mg/mL solution in water).

Tail vein injections of 250 μL of the complex were performed on ICR mice(n=3) using a 30 gauge, 0.5 inch needle. 30 min post injection, theanimals were injected with polyacrylic acid (1500 μg, 150 μL of a 10mg/mL solution in water). One day after injection, the animals weresacrificed, and a luciferase assay was conducted on the lung tissue. ALumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer wasused. The results are summarized in the table below.

Relative Luciferase Activity (light units) Complex n1 n2 n3 Complex I4,231,357 2,280,870 4,933,875 Complex II 2,671,040 2,525,587 1,186,365Complex III 1,774,820 878,562 2,969,813

The results indicate that the polyimidazolinium formulations deliverpDNA to the lung.

Example 8

Transfection of 3T3 Cells with Polyimidazolinium Formulations. SeveralComplexes were formed for cell transfections. Linear polyethyleniminewith N-propionyl substitution (10% of the amines acylated, 90%ethylamine by 1H NMR, Polysciences, Inc., lPEI10%/NPr was dissolved inwater to a concentration of 20 mg/mL. The pH of the solution wasadjusted to pH 7.5 with hydrochloric acid (12 M). The polyimidazoliniumderived from this polymer was also utilized in complex formation.

3T3 cells were maintained in DMEM. Approximately 24 h prior totransfection, cells were plated at an appropriate density in 48-wellplates and incubated overnight. Cultures were maintained in a humidifiedatmosphere containing 5% CO₂ at 37° C. The indicated amount of complexwas then combined with the cells in 1 mL media. Cells were harvestedafter 24 h and assayed for luciferase activity using a Lumat LB 9507(EG&G Berthold, Bad-Wildbad, Germany) luminometer. The amount ofluciferase expression was recorded in relative light units. Numbers arethe average for two separate wells.

W:W μL/ Average Sample complex Ratio Well RLU 1 pDNA lPEI10% NPr 5/5 40416,992 2 pDNA lPEI10% NPr 5/10 40 1,292,181 3 pDNA lPEI10% NPr 5/15 401,298,805 4 pDNA lPEI10% NPr 5/20 40 1,369,775 5 pDNA lPEI10% NPr 5/3040 1,511,175 6 pDNA lPEI10% NPr 5/40 40 1,067,322 7 pDNAPolyimidazolinium 5/5 40 155,842 8 pDNA Polyimidazolinium 5/10 40331,081 9 pDNA Polyimidazolinium 5/15 40 420,084 10 pDNAPolyimidazolinium 5/20 40 281,017 11 pDNA Polyimidazolinium 5/30 401,037,222 12 pDNA Polyimidazolinium 5/40 40 1,067,404

The results indicate that the polyimidazolinium is able to transfect 3T3cells at a lower efficiency than lPEI10% NPr under these conditions. ThelPEI10% NPr showed a similar toxicity profile (as judged by the finalconfluency) to the polyimidazolinium polymer.

Example 9

Transfection of HUH-7 Cells with Recharged PolyimidazoliniumFormulations. Linear polyethylenimine with N-propionyl substitution (10%of the amines acylated, 90% ethylamine by 1H NMR, Polysciences, Inc.),was dissolved in water to a concentration of 20 mg/mL. The pH of thesolution was adjusted to pH 7.5 with hydrochloric acid (12 M), and thepolyimidazolinium (PI) was formed at elevated temperature. The sampleswere recharged with various amounts of polyacrylic acid (10 mg/mLsolution in water).

HUH-7 cells were maintained in DMEM. Approximately 24 h prior totransfection, cells were plated at an appropriate density in 48-wellplates and incubated overnight. Cultures were maintained in a humidifiedatmosphere containing 5% CO2 at 37° C. The indicated amount of complexwas then combined with the cells in 1 mL of media. Cells were harvestedafter 24 h and assayed for luciferase activity using a Lumat LB 9507(EG&G Berthold, Bad-Wildbad, Germany) luminometer. The amount ofluciferase expression was recorded in relative light units. Numbers arethe average for two separate wells.

Transfection of HUH7 cell using DNA/polyimidazolinium (PI)/polyacrylicacid complexes. W:W μL/ Average Complex Ratio Well Media RLU pDNA/PI/PAA5/20/0 40 Opti 3981083 pDNA/PI/PAA 5/20/14 40 Opti 13111775 pDNA/PI/PAA5/20/16 40 Opti 12334305 pDNA/PI/PAA 5/20/18 40 Opti 11000786pDNA/PI/PAA 5/20/19 40 Opti 11458729 pDNA/PI/PAA 5/20/20 40 Opti10099756 pDNA/PI/PAA 5/20/21 40 Opti 7820030 pDNA/PI/PAA 5/20/22 40 Opti504225 pDNA/PI/PAA 5/20/0 40 Serum 2197 pDNA/PI/PAA 5/20/14 40 Serum544564 pDNA/PI/PAA 5/20/16 40 Serum 3165930 pDNA/PI/PAA 5/20/18 40 Serum8597630 pDNA/PI/PAA 5/20/19 40 Serum 7959588 pDNA/PI/PAA 5/20/20 40Serum 6975200 pDNA/PI/PAA 5/20/21 40 Serum 6504250 pDNA/PI/PAA 5/20/2240 Serum 36167

The results indicate that recharged polyimidazolinium formulations areeffective in transfecting HUH-7 cells.

Example 10

Transfection of HUH-7 Cells with Binary and Ternary PolyimidazoliniumFormulations. Linear polyethylenimine with N-propionyl substitution(lPEI10% NPr, 10% of the amines acylated, 90% ethylamine by 1H NMR,Polysciences, Inc.), was dissolved in water to a concentration of 20mg/mL. The pH of the solution was adjusted to pH 7.5 with hydrochloricacid (12 M). A portion of this solution was used to form thepolyimidazolinium (PI) by holding the solution at elevated temperature.The samples were recharged with various amounts of polyacrylic acid (10mg/mL solution in water). HUH-7 cells were maintained in DMEM.Approximately 24 h prior to transfection, cells were plated at anappropriate density in 48-well plates and incubated overnight. Cultureswere maintained in a humidified atmosphere containing 5% CO2 at 37° C.The indicated amount of complex was then combined with the cells in 1 mLof media. Cells were harvested after 24 h and assayed for luciferaseactivity using a Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer. The amount of luciferase expression was recorded inrelative light units. Numbers are the average for two separate wells.

W:W μL/ Average Final complex Ratio Well RLU Confluency: DNA/PI/PAA5/10/0 40 4292994 100 DNA/PI/PAA 5/20/0 40 3282018 85 DNA/PI/PAA 5/40/040 3477386 85 DNA/PI/PAA 5/60/0 40 3233045 75 DNA/PI/PAA 5/40/25 408530972 75 DNA/PI/PAA 5/40/30 40 7066358 75 DNA/PI/PAA 5/40/35 406675965 85 DNA/PI/PAA 5/40/40 40 6669150 85 DNA/lPEI10% NPR/PAA 5/10/040 1895920 90 DNA/lPEI10% NPR/PAA 5/20/0 40 3881579 85 DNA/lPEI10%NPR/PAA 5/40/0 40 5655288 60 DNA/lPEI10% NPR/PAA 5/60/0 40 431360 60DNA/lPEI10% NPR/PAA 5/40/25 40 2390180 60 DNA/lPEI10% NPR/PAA 5/40/30 406709513 60 DNA/lPEI10% NPR/PAA 5/40/35 40 8483672 65 DNA/lPEI10% NPR/PAA5/40/40 40 7779514 65

The results indicate that the polyimidazolinium polymer is effective attransfecting HUH-7 cells either as a binary or a ternary, rechargedcomplex. The results indicate that the recharged complex is moreeffective that the corresponding particle on the non-cyclized polymer.

Example 11

Methylation of 2-Methyl-2-Imidazoline. To a solution of2-methyl-2-imidazoline (52.7 mg, 0.626 mmol, Aldrich Chemical Company)in 3 mL acetonitrile was added methyl iodide (86 μL, 1.377 mmol, AldrichChemical Company). The resulting solution was heated to reflux. After 3days, the solution was removed from heat, and diethyl ether was added toprecipitate the salts. The resulting precipitate was dried under vacuum.The material was analyzed by mass spectroscopy (Sciex API 150EX) andindicated a mixture of 1,2-dimethyl imidazolinium iodide (M=99),1,2,3-trimethyl imidazolinium iodide (M=113), and 1,1,4-trimethyl,4-acyl-ethylenediamine (M+1=145). The mixture was analyzed by UVspectroscopy (Beckman DU530 Spectrophotometer), and indicated a shift inλmax from 214 nm to 224 nm for the imidazolinium iodide sample relativeto the starting imidazoline.

Example 12

Polymerization of 2-Methyl-2-Imidazoline and 1,4-Butanediol DiglycidylEther. To a solution of 2-methyl-2-imidazoline (200 mg, 2.38 mmol,Aldrich Chemical Company) in 5 mL DMF was added 1,4-Butanedioldiglycidyl ether (458 μL, 2.38 mmol, Aldrich Chemical Company). Thesolution was heated at reflux for 1.5 h, and the resulting polymer gelwas washed with diethylether. The mixture was analyzed by UVspectroscopy (Beckman DU530 Spectrophotometer), and indicated a shoulderabsorbance of λ235 nm for the imidazolinium polymer, indicatingincorporation of the ring system into the polymer.

Example 13

Polymerization of 2-Methyl-2-Imidazoline and Epichlorohydrine. To asolution of 2-methyl-2-imidazoline (200 mg, 2.38 mmol, Aldrich ChemicalCompany) in 5 mL DMF was added epichlorohydrine (186 μL, 2.38 mmol,Aldrich Chemical Company). The solution was heated at reflux for 16 h,and the resulting polymer was precipitated with diethylether. Theprecipitate was analyzed by UV spectroscopy (Beckman DU530Spectrophotometer), and indicated an absorbance of λ235 nm for theimidazolinium polymer, indicating incorporation of the ring system intothe polymer.

Example 14

Polymerization of 2-Methyl-2-Imidazoline and Polyepichlorohydrine. To asolution of polyepichlorohydrine (102.9 mg, 1.48 mmol in Cl) in 2 mLacetonitrile, was added 2-methyl-2-imidazoline (125 mg, 1.48 mmol,Aldrich Chemical Company). The solution was heated at reflux for 16 h,and the resulting polymer was precipitated with diethylether. Theprecipitate was analyzed by UV spectroscopy (Beckman DU530Spectrophotometer), and indicated a new shoulder absorbance of λ220 nmfor the imidazolinium polymer, indicating incorporation of the ringsystem into the polymer.

Example 15 Partial Deprotections of 2-Ethyl-2-Oxazoline

A. To a solution of 2-ethyl-2-oxazoline (5.00 g, 50.4 mmol in amide,Aldrich Chemical Company) in 40 mL of water was added HCl (conc, 2.05mL, 25.2 mmol, 0.5 eq) dropwise over several minutes. The resultingsolution was heated at reflux for 56 h. The pH was neutralized to pH 7with NaOH (12 M). The solution was concentrated under reduced pressureand taken up in EtOH and filtered to remove NaCl. The ethanol filtrationwas repeated 5 times to afford a copolymer of the following subunits:ethyl amine, 2-ethylimidazolinium, and N-propionyl-ethyl amine. Theratios of the subunits were determined by 1H NMR to be 88.9%, 4.9%, and6.2% respectively.

B. To a solution of 2-ethyl-2-oxazoline (5.00 g, 50.4 mmol in amide,Aldrich Chemical Company) in 40 mL of water was added HCl (con, 2.67 mL,32.8 mmol, 0.65 eq) dropwise over several minutes. The resultingsolution was heated at reflux for 56 h. The pH was neutralized to pH 7with NaOH (12 M). The solution was concentrated under reduced pressureand taken up in EtOH and filtered to remove NaCl. The ethanol filtrationwas repeated 5 times to afford a copolymer of the following subunits:ethyl amine, 2-ethylimidazolinium, and N-propionyl-ethyl amine. Theratios of the subunits were determined by 1H NMR to be 92.8%, 4%, and3.2% respectively.

C. To a solution of 2-ethyl-2-oxazoline (5.00 g, 50.4 mmol in amide,Aldrich Chemical Company) in 40 mL of water was added HCl (con, 3.48 mL,42.8 mmol, 0.85 eq) dropwise over several minutes. The resultingsolution was heated at reflux for 56 h. The pH was neutralized to pH 7with NaOH (12 M). The solution was concentrated under reduced pressureand taken up in EtOH and filtered to remove NaCl. The ethanol filtrationwas repeated 5 times to afford a copolymer of the following subunits:ethyl amine, 2-ethylimidazolinium, and N-propionyl-ethyl amine. Theratios of the subunits were determined by 1H NMR to be 95%, 3.35%, and1.65% respectively.

D. To a solution of 2-ethyl-2-oxazoline (5.00 g, 50.4 mmol in amide,Aldrich Chemical Company) in 40 mL of water was added HCl (con, 6.14 mL,75.6 mmol, 1.5 eq) dropwise over several minutes. The resulting solutionwas heated at reflux for 48 h. The pH was neutralized, than made basic,to pH 14 with NaOH (12 M). The solution was heated at reflux for 16 h.After 16 h, the pH was neutralized to pH 7 with HCl (con). The solutionwas concentrated under reduced pressure and taken up in EtOH andfiltered to remove NaCl. The ethanol filtration was repeated 5 times toafford a copolymer of the following subunits: ethyl amine,2-ethylimidazolinium, and N-propionyl-ethyl amine. The ratios of thesubunits were determined by 1H NMR to be 100%, 0%, and 0% respectively.

E. To a solution of 2-ethyl-2-oxazoline (5.00 g, 50.4 mmol in amide,Aldrich Chemical Company) in 40 mL of water was added NaOH (3.024 g,75.6 mmol, Aldrich Chemical Company) portion wise over several minutes.The polymer started to drop out as the pH increased. The resultingsuspension was heated at reflux for 56 h. The pH was neutralized to pH 7with HCl (con). The solution was concentrated under reduced pressure andtaken up in EtOH and filtered to remove NaCl. The ethanol filtration wasrepeated 5 times to afford a copolymer of the following subunits: ethylamine, 2-ethylimidazolinium, and N-propionyl-ethyl amine. The ratios ofthe subunits were determined by 1H NMR to be a trace %, a trace %, and98% respectively.

Example 16

Acylation of Polyvinyl Amine and the Formation of an Amidinium Ring. Toa solution of polyvinyl amine hydrochloride (40 mg, 0.503 mmol in amine,25 K, Aldrich Chemical Company) in 400 μL water was addeddiisopropylethylamine (35 μL, 1.00 mmol, Aldrich Chemical Company),followed by acetic anhydride (9.6 μL, 0.503 mmol, Aldrich ChemicalCompany). The resulting solution was stirred for 12 h, and split intotwo equal sized portions. Sample 1 (MC 1025) was heated at 70° C. for 16h. Sample 2 (MC1026) was stored at 4° C.

Example 17

Acylation of Polyallylamine and the Formation of an Amidinium Ring. To asolution of polyallylamine hydrochloride (40 mg, 0.428 mmol in amine, 15K, Aldrich Chemical Company) in 400 μL water was addeddiisopropylethylamine (30 μL, 0.855 mmol, Aldrich Chemical Company),followed by acetic anhydride (8.1 μL, 0.428 mmol, Aldrich ChemicalCompany). The resulting solution was stirred for 12 h, and split intotwo equal sized portions. Sample 1 (MC 1029) was heated at 70° C. for 16h. Sample 2 (MC1030) was stored at 4° C.

Example 18

Delivery of siRNA to Cells In Vitro. Several complexes were prepared forin vitro transfection of 3T3-Luc cell. Some formulations include a lipid(MC 798), while others are lipid free. Transfections were conducted in10% serum. 3T3-Luc cells were maintained in DMEM. Approximately 24 hprior to transfection, cells were plated at an appropriate density in48-well plates and incubated overnight. Cultures were maintained in ahumidified atmosphere containing 5% CO₂ at 37° C. The indicated amountof complex containing anti-luciferase siRNA (GL-2) and the indicatedpolymer was then combined with the cells in 1 mL of media. Cells wereharvested after 24 h and assayed for luciferase activity using a LumatLB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer. The amount ofluciferase expression was recorded in relative light units. Numbers arethe average for two separate wells.

Cell type: 3T3-Luc

Concentration μL/ Sample Formulation w:w ratio Well RLU Blank X 1002100570 GL-2 X 200 100 1976795 GL-2 TKO 200/8 uL 100 1094425 GL-2 TKO200 ng/12 μL 100 517800 GL-2 PVA control 200 ng/6 μL 100 1952695 GL-21025 200 ng/6 μL 100 1480135 GL-2 1026 200 ng/6 μL 100 1539960 GL-2 PAAcontrol 200 ng/6 μL 100 1711830 GL-2 1029 200 ng/6 μL 100 1554965 GL-21030 200 ng/6 μL 100 1585145 GL-2/798 PVA control 200 ng/4 μg/6 μL 1001829290 GL-2/798 1025 200 ng/4 μg/6 μL 100 1432655 GL-2/798 1026 200ng/4 μg/6 μL 100 1511445 GL-2/798 PAA control 200 ng/4 μg/6 μL 1001610610 GL-2/798 1029 200 ng/4 μg/6 μL 100 1404530 GL-2/798 1030 200ng/4 μg/6 μL 100 1840955

The results indicate that the siRNA can be delivered to cells using theindicated polymers thus inhibiting expression of the luciferase gene.

Example 19

Delivery of DNA to Cells In Vitro. Several complexes were prepared forin vitro transfection of HEPA cells. The formulations include a lipid(MC 798). Transfections were conducted in 10% serum. HEPA cells weremaintained in DMEM. Approximately 24 h prior to transfection, cells wereplated at an appropriate density in 48-well plates and incubatedovernight. Cultures were maintained in a humidified atmospherecontaining 5% CO₂ at 37° C. The indicated amount of complex was thencombined with the cells in 1 mL of media. Cells were harvested after 24h and assayed for luciferase activity using a Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer. The amount of luciferaseexpression was recorded in relative light units. Numbers are the averagefor two separate wells.

Cell type: HEPA

concentration w:w:w ratio polymer (DNA/lipid/polymer) RLU pAA 3/9/6347944 pAA 3/15/6 1060816 1029 3/9/6 2942677 1029 3/15/6 7128621 10303/9/6 2857683 1030 3/15/6 2821997 1029 3/9/12 2597371The results indicate that the polyamidinium polymer is able to transferDNA to cells in vitro.

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

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1. A composition for delivering a polynucleotide to a cell comprising: acationic poly(cyclic amidinium)-containing compound capable ofcondensing nucleic acid and the polynucleotide.
 2. The composition ofclaim 2 wherein the poly cyclic amidinium consists of a poly1,3-piperazinium.
 3. The composition of claim 2 wherein thepolynucleotide is selected from the group consists of: DNA, RNA, andsiRNA.
 4. The composition of claim 2 wherein the poly cyclic amidiniumis selected from the group consisting of: partially acylatedpolyethylenimine, partially acylated polyallylamine, and partiallyacylated polyvinylamine.
 5. The composition of claim 2 wherein the polycyclic amidinium consists of a poly 1,3-nitrogen-8-membered heterocyclicring.
 6. The composition of claim 5 wherein the polynucleotide isselected from the group consists of: DNA, RNA, and siRNA.
 7. Thecomposition of claim 2 wherein the poly cyclic amidinium is selectedfrom the group consisting of: partially acylated polyethylenimine,partially acylated polyallylamine, and partially acylatedpolyvinylamine.
 8. The composition of claim 1 wherein the cell consistsof an in vivo cell.
 9. The composition of claim 11 wherein the in vivocell consists of a lung cell.
 10. The composition of claim 11 whereinthe in vivo cell consists of a liver cell.
 11. The composition of claim13 wherein the liver cell consists of a hepatocyte.